Chang Jin Park

Induction of pepper cDNA encoding a lipid transfer protein during the resistance response to tobacco mosaic virus

Pepper (Capsicum annuum) plants exhibit hypersensitive response (HR) against infection by many tobamoviruses. A clone encoding a putative nonspecific lipid transfer protein (CaLTP1) was isolated by differential screening of a cDNA library from resistant pepper leaves when inoculated with tobacco mosaic virus (TMV) pathotype P0. The predicted amino acid sequence of CaLTP1 is highly similar to that of the other plant LTPs. Southern blot analysis showed that a small gene family of LTP-related sequences was present in the pepper genome. Transcripts homologous to CaLTP1 accumulated abundantly in old leaves and flowers. CaLTP1 expression was induced in the incompatible interaction with TMV-P0 but was not induced in the compatible interaction with TMV-P1.2. In correlation with the temporal progression of HR in the inoculated leaves, CaLTP1 transcripts started to accumulate at 24 h after TMV-P0 inoculation, reaching a maximal level at 48 h. A strain of Xanthomonas campestris pv. vesicatoria (Xcv) that carries the bacterial avirulence gene, avrBs2, was infiltrated into leaves of a pepper cultivar containing the Bs2 resistance gene. A marked induction of CaLTP1 expression was observed in Xcv-infiltrated leaves. Effects of exogenously applied abiotic elicitors on CaLTP1 expression were also examined. Salicylic acid caused a rapid accumulation of CaLTP1 transcripts in pepper leaves and ethephon treatment also induced the expression of the CaLTP1 gene. Transient expression in the detached pepper leaves by biolistic gene bombardment indicated that CaLTP1 is localized mostly at the plant cell surface, possibly in the cell wall. These results suggest possible role(s) for LTPs in plant defense against pathogens including viruses.

Isolation of pepper mRNAs differentially expressed during the hypersensitive response to tobacco mosaic virus and characterization of a proteinase inhibitor gene

Abstract Pepper ( Capsicum annuum L. cv. Bugang) displays a hypersensitive response (HR) upon infection by tobacco mosaic virus (TMV) pathotype P 0 . To elucidate molecular mechanisms underlying this response, a cDNA library was constructed and differentially screened between TMV-P 0 -inoculated and uninoculated leaves. Of 3260 clones analyzed by reverse RNA slot-blot hybridization, 22 showed up-regulated expression upon TMV-P 0 challenge. Among these, 16 clones showed significantly higher levels of expression in response to the avirulent pathotype TMV-P 0 than to the virulent pathotype TMV-P 1.2 . Nucleotide sequence analysis and database searches revealed that the polypeptides deduced from the selected clones displayed significant sequence homology to various known proteins, indicating conservation of the wide range of up-regulated proteins during HR against viral challenge in higher plants. A cDNA clone encoding a putative proteinase inhibitor II ( CaPinII ) was selected and further analyzed. Upon TMV-P 0 inoculation, the CaPinII transcript accumulated maximally at 72 h post-inoculation and declined afterwards. In contrast, inoculation with TMV-P 1.2 did not induce CaPinII expression to any detectable level. The CaPinII gene was also expressed in response to the treatment of various chemicals, as well as wounding. Southern blot analysis showed that a small family of genes homologous to the CaPinII gene is present in the pepper genome.

A hot pepper gene encoding WRKY transcription factor is induced during hypersensitive response to Tobacco mosaic virus and Xanthomonas campestris

Plant WRKY transcription factors were previously implicated in the alteration of gene expression in response to various pathogens. The WRKY proteins constitute a large family of plant transcription factors, whose precise functions have yet to be elucidated. Using a domain-specific differential display procedure, we isolated a WRKY gene, which is rapidly induced during an incompatible interaction between hot pepper and Tobacco mosaic virus (TMV) or Xanthomonas campestris pv . vesicatoria (Xcv). The full-length cDNA of CaWRKY-a (Capsicum annuum WRKY-a) encodes a putative polypeptide of 546 amino acids, containing two WRKY domains with a zinc finger motif. The expression of CaWRKY-a could be rapidly induced by not only chemical elicitor such as salicylic acid (SA) or ethephon but also wounding treatments. The nuclear localization of CaWRKY-a was determined in transient expression system using tobacco BY-2 cells by polyethylene glycol (PEG)-mediated transformation experiment. With oligonucleotide molecules containing the putative W-box sequences as a probe, we confirmed that CaWRKY-a protein had W-box-binding activity. These results suggest that CaWRKY-a might be involved as a transcription factor in plant defense-related signal transduction pathways.

Capsicum annuum WRKYb transcription factor that binds to the CaPR-10 promoter functions as a positive regulator in innate immunity upon TMV infection.

In plant, some WRKY transcription factors are known to play an important role in the transcriptional reprogramming associated with the immune response. By using WRKY-domain-specific differential display procedure, we isolated CaWRKYb gene, which is rapidly induced during an incompatible interaction between hot pepper and Tobacco mosaic virus (TMV) pathotype P(0) infection. The recombinant CaWRKYb bound to the W box-containing CaPR-10 promoter probes efficiently and the specificity of binding was confirmed by mutant study and competition with cold oligonucleotides. Also, in GUS reporter activity assay using Arabidopsis protoplasts with the CaPR-10 promoter, GUS activity was increased in the presence of CaWRKYb. And CaWRKYb-knockdown plant showed reduced number of hypersensitive response local lesions upon TMV-P(0) infection. Furthermore, CaWRKYb-knockdown plant exhibited compromised resistance to TMV-P(0) by accumulating more TMV, apparently through decreased expression of CaPR-10, CaPR-1, and CaPR-5. These results suggest that CaWRKYb is involved as a positive transcription factor in defense-related signal transduction pathways in hot pepper.

CaAlaAT1 catalyzes the alanine: 2-oxoglutarate aminotransferase reaction during the resistance response against Tobacco mosaic virus in hot pepper

Hot pepper (Capsicum annuum L. cv. Bugang) plants exhibit a hypersensitive response (HR) upon infection by Tobacco mosaic virus (TMV) pathotype P0. To elucidate molecular mechanism that underlies this resistance, hot pepper cv. Bugang leaves were inoculated with TMV-P0 and genes specifically up-regulated during the HR were isolated by differential screening. One of the clones, CaAlaAT1 encoding a putative alanine aminotransferase (EC 2.6.1.2) exhibited organ-specific expression pattern and the transcript accumulated abundantly in red (ripe) fruit tissues. CaAlaAT1 transcript was also induced in older leaves during senescence. The expression of CaAlaAT1 gene was increased in the incompatible interaction with TMV-P0 but was not in the compatible interaction with TMV-P1.2. When a strain of Xanthomonas campestris pv. vesicatoria (Xcv) carrying an AvrBs2 gene was infiltrated into the leaves of a pepper cv. ECW 20R carrying Bs2 resistance gene, a marked induction and maintenance of CaAlaAT1 gene expression was observed. The expression of CaAlaAT1 gene was triggered by salicylic acid (SA) and ethylene but not by methyl jasmonate (MeJA). CaAlaAT1 seemed to be localized mostly at the cytosol from the polyethylene glycol (PEG)-mediated transformation experiment. CaAlaAT1 seemed to catalyze alanine: 2-oxoglutarate aminotransferase (AKT) reaction, which was a main activity among the four activities in vitro, during the resistance response against TMV in hot pepper. These results suggest that CaAlaAT1, a protein known to be involved in metabolic reactions, might be one of the components in the plant’s defense signal pathway against pathogens.

Induction of a cytosolic pyruvate kinase 1 gene during the resistance response to Tobacco mosaic virus in Capsicum annuum

Hot pepper (Capsicum annuum L. cv. Bugang) plants exhibit a hypersensitive response (HR) upon infection by Tobacco mosaic virus (TMV) pathotype P0. Previously, to elucidate molecular mechanism that underlies this resistance, hot pepper cv. Bugang leaves were inoculated with TMV-P0 and genes specifically up-regulated during the HR were isolated by microarray analysis. One of the clones, Capsicum annuum cytosolic pyruvate kinase 1 (CaPKc1) gene was increased specifically in the incompatible interaction with TMV-P0. The expression of CaPKc1 gene was also triggered not only by various hormones such as salicylic acid (SA), ethylene, and methyl jasmonate (MeJA), but also NaCl and wounding. These results suggest that CaPKc1 responds to several defense-related abiotic stresses in addition to TMV infection.

Capsicum annuum tobacco mosaic virus-induced clone 1 expression perturbation alters the plant's response to ethylene and interferes with the redox homeostasis

Capsicum annuum tobacco mosaic virus (TMV)-induced clone 1 (CaTin1) gene was expressed early during incompatible interaction of hot pepper (Caspsicum annuum) plants with TMV and Xanthomonas campestris. RNA-blot analysis showed that CaTin1 gene was expressed only in roots in untreated plants and induced mainly in leaf in response to ethylene, NaCl, and methyl viologen but not by salicylic acid and methyl jasmonate. The ethylene dependence of CaTin1 induction upon TMV inoculation was demonstrated by the decrease of CaTin1 expression in response to several inhibitors of ethylene biosynthesis or its action. Transgenic tobacco (Nicotiana tabacum) plants expressing CaTin1 gene in sense- or antisense-orientation showed interesting characteristics such as the accelerated growth and the enhanced resistance to biotic as well as abiotic stresses. Such characteristics appear to be caused by the elevated level of ethylene and H2O2. Moreover, in transgenic plants expressing antisense CaTin1 gene, the expression of some pathogenesis-related genes was enhanced constitutively, which may be mainly due to the increased ethylene level. The promoter of CaTin1 has four GCC-boxes, two AT-rich regions, and an elicitor-inducible W-box. The induction of the promoter activity by ethylene depends on GCC-boxes and by TMV on W-box. Taken together, we propose that the CaTin1 up-regulation or down-regulation interferes with the redox balance of plants leading to the altered response to ethylene and biotic as well as abiotic stresses.

Erratum: Induction of a pepper cDNA encoding SAR8.2 protein during the resistance response to tobacco mosaic virus (Molecules and Cells (2001) 12 (250-256)

Retraction Note: Mol. Cells 12 (2001) 250–256 Members of the editorial board have unanimously agreed to retract the article [Mol. Cells 12 (2001) 250–256] for potential misconducts mainly concerning manipulation and repeated uses of photomicrographs of control data internally along with mislabeling and/or externally in multiple publications. As specified in the “Instructions to Authors”, Molecules and Cells (Mol. Cells) explicitly prohibits mis-representation or falsification of experimental data including duplication of previously published data. In the article, lanes 1-4 and lanes 5–7 of rRNA gels in Fig. 2B are identical to lanes 1–4 and lanes 10–12 in Fig. 3A, respectively; two EtBr gels of rRNA in Figs. 4A and 4B are identical, and rRNA gels in Figs. 5A, 5B, and 5C are identical as well.

Involvement of the Fas-associated Factor1 Ortholog, CaFAF1, in Regulating Programmed Cell Death in Plants

Programmed cell death (PCD) in plant cells is often accompanied by biochemical and morphological hallmarks similar to those of animal apoptosis. However, orthologs of several core components of such apoptosis have not been reported in plants. Here, we describe an ortholog of Fas-associated factor1 (FAF1), a member of the Fas-death-inducing signaling complex (Fas-DISC), found in pepper. We also examined FAF1 orthologs in other plant species. Transcripts of CaFAF1 specifically accumulated in pepper leaves infected with the avirulent pepper mild mottle virus (PMMoV) P0 pathotype. This gene was also strongly expressed in aging leaves. To determine whether those orthologs are involved in PCD, we suppressed their expression through virus-induced gene silencing, and determined the effect on the hypersensitive response (HR), a typical PCD, in pepper, tomato, and tobacco. Constitutive expression of CaFAF1 in transgenic tobacco triggered spontaneous induction of cell death lesions and induced pathogenesis-related (PR) genes. This ability to cause cell death and suppress R gene-mediated HR in its knockdown condition suggests that some features of animal and plant cell death processes may be shared. We propose that plant FAF1 is a conserved cell death regulator in both kingdoms.

Erratum: A hot pepper cDNA encoding ascorbate peroxidase is induced during the incompatible interaction with virus and bacteria (Molecules and Cells (2002) 14 (75-84)

Retraction Note: Mol. Cells 14 (2002) 75–84 Members of the editorial board have unanimously agreed to retract the article [Mol. Cells 14 (2002) 75–84] for potential misconducts mainly concerning manipulation and repeated uses of photomicrographs of control data internally along with mislabeling and/or externally in multiple publications. As specified in the “Instructions to Authors”, Molecules and Cells (Mol. Cells) explicitly prohibits mis-representation or falsification of experimental data including duplication of previously published data. In the article, lanes 1-6 and Lanes 10–15 of EtBr gel of rRNA in Fig. 5 are mirror images of each other; a part of this image has been previously used in Fig. 4B of Mol. Cells 11 (2001) 122–127; this image has also been used in Fig. 2D of Plant Physiol. 135 (2004) 561–573; rRNA gels in Figs. 6A, 6B, and 6C are identical, and rRNA gels in Figs. 7A and 7B are identical as well.